The field of the invention relates to biosensors comprising recombinant proteins and reporter systems. In particular, the field of the invention relates to biosensors comprising recombinant proteins that bind to a ligand, such as a cellular metabolite, and then modulate transcription of a reporter based on binding to the ligand.
Efforts to engineer microbial factories have benefitted from mining biological diversity and high throughput synthesis of novel enzymatic ensembles, yet screening and optimizing metabolic pathways remain rate-limiting steps. Metabolite-responsive biosensors may help to address these persistent challenges by enabling the monitoring of metabolite levels in individual cells and the implementation of metabolite-responsive feedback control. We are currently limited to naturally-evolved biosensors, which are insufficient for monitoring many metabolites of interest. Thus, a method for engineering novel biosensors would be powerful, yet we lack a generalizable approach that enables the construction of a wide range of biosensors. As a step towards this goal, we developed a bottom-up strategy for converting metabolite-binding proteins into metabolite-responsive transcriptional regulators. By pairing a modular protein design approach with a library of synthetic promoters and applying robust statistical analyses, we identified quantitative design principles for engineering biosensor-regulated promoters and for achieving design-driven improvements of biosensor performance. We demonstrated the feasibility of this strategy by fusing a programmable DNA binding motif (zinc finger module) with a model ligand binding protein (maltose binding protein), to generate a novel biosensor conferring maltose-regulated gene expression. This technology enables the design of novel biosensors for diverse synthetic biology applications.